OBJECTIVETo explore the preliminary methods of in vitro isolation, culture and identification of sebocytes and eccrine sweat gland cells from human fetal skin.

METHODSHuman fetal skin was digested with dispase or type II collagenase, and then by micro - sieving to isolate human sebaceous gland and eccrine sweat gland cells. DMEM/F12 (1: 1) was used as the basic culture medium, supplemented with fetal bovine serum, recombinant human epidermal growth factor, L-glutamine, Hydrocortisone, choleratoxin, penicillin and streptomycin as the medium for sebocytes, or fetal bovine serum, recombinant human epidermal growth factor, triiodothyronine, hydrocortisone, insulin, transferrin, sodium selenite to the medium for eccrine sweat gland duct cells. Primary cultures and subcultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2/95% oxygen. Cell morphology was observed by inverted phase contrast microscopy, and the cultured cells were identified with cell clone efficiency determination. The cultured sebocytes were identified with oil red staining and CK4.62, Epithelia Membrane Antigen (EMA) immunohistochemistry staining. The cultured eccrine sweat gland duct cells were identified with CK7, CK19 immunohistochemistry staining.

RESULTSThe isolated sebocytes and eccrine sweat gland cells from human fetal skin could grow by adhering to the wall and proliferate in vitro. The cell clone efficiency of human fetal sebocytes was 2.7%, which was obviously lower than that of human fetal keratinocytes (8.0%, P < 0.01). There was no obvious difference in the cell clone efficiency between human fetal eccrine sweat gland cells (7.3%) and human fetal keratinocytes (7.7%, P > 0.05) . The results of oil red staining indicated that a small quantity of lipid droplets in sebocytes, and immunohistochemistry staining of CK4.62, EMA were positive in subculture sebocytes. The immunohistochemistry staining of CK7, CK19 was positive in subculture eccrine sweat gland duct cells.

CONCLUSIONIn vitro cultured human fetal sebocytes and eccrine sweat gland duct cells displayed the markers and biological characteristics of epithelial lineage, but human fetal sebocytes proliferated more